High quality, pure crystals are of great value for a variety of industrial and research applications. For example, a frequent problem in pharmaceutical research is the difficulty of obtaining large single crystals for X-ray structural analyses to determine molecular conformations. Crystallization is also frequently used as a method of purification in the biotechnology and pharmaceutical industries. More highly ordered crystals will typically result in a more highly purified product. Larger crystals can also impart advantages in packaging and handling operations.
Crystallization is conventionally carried out from the melt, from vapor, or from solution. The first two methods are generally restricted to materials that are thermally stable at high temperatures. Almost all biologically interesting molecules are crystallized from solution. Crystal growth from a solution involves shifting the solid versus solution equilibrium so that the solution becomes supersaturated with the substance to be crystallized. The most common methods of shifting the equilibrium are thermal and chemical. Thermally shifting the equilibrium between solid and solution relies on the temperature dependence of solubility to promote crystallization. Equilibrium of solid and solution can also be chemically shifted, by the addition of a second solvent which has good miscibility with the first solvent but in which the solute is less soluble. The addition of this secondary solvent, known as an anti-solvent, will reduce the solubility of the solute, thus causing it to precipitate.
Over the past decade, compressed gases, liquefied gases, and materials intermediate to gases and liquids known as supercritical fluids have been used as anti-solvents. A pure compound becomes critical at its critical temperature (T.sub.c) and critical pressure (P.sub.c). A compound becomes a supercritical fluid above its critical temperature and at its critical pressure, or above its critical pressure and at its critical temperature, or where conditions exceed both the critical temperature and pressure. These parameters are intrinsic thermodynamic properties of all sufficiently stable pure component compounds. Carbon dioxide, for example, becomes a supercritical fluid at conditions equal to or exceeding its critical temperature of 31.1.degree. C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical region, normally gaseous compounds exhibit greatly enhanced solvation power. At a pressure of 3,000 psig (204 atm) and a temperature of 40.degree. C., carbon dioxide has a density around 0.8 g/ml and behaves very much like a nonpolar organic solvent, with a zero dipole moment.
Compounds which are capable of forming a critical fluid undergo a transition as such compounds approach critical temperatures and pressures. Within approximately .+-.25% Kelvin of the critical temperature and within approximately .+-.25% of the critical pressure, compounds exhibit fluid density and solvation properties which approach those of critical and supercritical fluids. These compounds, under such conditions approaching critical conditions, are commonly referred to as near critical fluids.
Recently, numerous compounds showing various antineoplastic and other bioactivities have been screened and identified from plant materials, microorganisms and marine organisms. Useful compounds found by these and other screening processes often require large and pure crystals in order to efficiently conduct structural analysis. Structural analysis is often helpful to better understand the mechanisms that are responsible for their specific therapeutic efficacy. Investigation of these compounds for their bioactivities has been hampered by their low natural abundance and the characteristics of conventional purification and crystallization processes. Paclitaxel (NSC 125973, better known as taxol.RTM., a trademark of the Bristol-Myers Squibb Company), an anticancer drug originally derived from the western yew Taxus brevifolia, is an example of such a compound. This material typically gives needle-like crystals from conventional crystallization (Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P. and McPhail, A. T., Plant Antitumor Agents. VI. Isolation and Structure of Taxol, A Novel Antileukemic and Antitumor Agent From Taxus brevifolia, Journal of American Chemical Society, 93:2325-2327, 1971).
Paclitaxel is a moderately sized molecule (MW=854, 113 atoms) with a complex structure that may hinder crystallization by conventional means or otherwise. As used herein, the term complex refers to molecules having approximately twenty-five or more atoms, and which are not comprised entirely of repeating subunits. For example, proteins are considered to have complex structure but polyethylene is not. The structure of paclitaxel is represented by the formula below: ##STR1##
Many other natural therapeutics of current interest likewise possess complex structures, e.g. bryostatin 1 (MW=905, 132 atoms). The structure of bryostatin 1 is represented by the formula below: ##STR2##
A need exists for a process which can produce large, highly ordered crystals of complex molecules.